Effect of anticoagulants on the activity of trypsin-like proteinases in human blood plasma

Influence of some anticoagulants (heparin, sodium citrate, their mixture) on blood trypsin-like proteinases activity was examined. The activity was determined using synthetic substrate N-alpha-benzoyl-L-arginine-p-nitroanilide. It was shown that heparin greatly activated blood trypsin-like proteinases (at heparin concentration 5 unit/ml of blood, the enzyme activity in plasma was about 10 times higher than the activity in serum). Heparin added to serum caused the activation effect too, maximum of activation was reached at heparin concentration in serum 800 unit/ml, following increase of heparin concentration did not led to the activity change. Sodium citrate had no significant effect both on the trypsin-like proteinases activity and on the activation effect of heparin. It was found that investigated anticoagulants did not affect blood antitryptic activity.     

Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.     

Effect of N-3-methyladenine on peptidase and ribonuclease activity of proteasome

Using a culture of cardiomyocytes it has been shown, that a well-known inhibitor of autophagy, N-3-methyladenine causes a 1.4 fold increase (p = 0.023) of the chymotrypsin-like activity, a 1.5 fold increase (p = 0.09) of the peptidyl-glutamyl peptide-hydrolyzing activity and 1.5 fold decrease (p = 0.07) of the trypsin-like activity of the proteasome. N-3-methyladenine in a dose-dependent manner inhibits chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the purified 20S proteasome, but activates it trypsin-like activity. Chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the 26S proteasome from proteasome fraction II did change in the same way, as in the case of 20S proteasome, but trypsin-like activity decreased. Using the above method of determining ribonuclease activity, we have shown, that N-3-methyladenine and clasto-lactacystin b-lactone inhibit the RNase activity of the proteasome. Specific proteasome inhibitor exhibits more powerful action, almost completely preventing RNA of actin and myosin from degradation. These data show a multitarget action of N-3-methyladenine, resulting in changes of peptidase and ribonuclease activity of the proteasome.     

Induction of the acrosome reaction in black tiger shrimp (Penaeus monodon) requires sperm trypsin-like enzyme activity

Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45-60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.     

Trypanocidal activity of peptidyl vinyl ester derivatives selective for inhibition of mammalian proteasome trypsin-like activity

Nine vinyl ester tripeptides selective for inhibition of mammalian proteasome trypsin-like activity were tested for in vitro activity against Trypanosoma brucei. Interestingly, two compounds showed trypanocidal activity in the low micromolar range without displaying cytotoxicity against human cells. However, the compounds did not inhibit the trypsin-like activity of the trypanosome proteasome although their effect correlates with inactivation of the chymotrypsin-like activity. This finding shows that the inhibitor sensitivities between mammalian and trypanosome proteasome are distinct. This difference may be exploited for rational anti-trypanosomal drug development.     

Trypsin-like activity in the vaginal epithelial cells of the rat.

Rat vaginal epithelial cells have trypsin-like activity as shown by the formation of a colored product when the cells are incubated with alpha-N-methyl alpha-N-toxyl-L-lysine beta-naphthol ester and hexazotized pararosanilin. This enzyme activity in vaginal smears is maximal at proestrus, i.e., the day in the 5-day estrus cycle when plasma estrogen is maximal. Only the rounded nucleated epithelial cells present at late diestrus, proestrus and early estrus demonstrate the trypsin-like enzyme activity. These are the cells that stain blue in the Papanicolaou method. Preincubation of cell suspensions with the serine protease inhibitor, p-nitrophenyl p-guanidino benzoate, prevented the enzyme staining reaction, further demonstrating the trypsin-like nature of the cellular enzyme. The advantages of this enzyme staining technique over the fibrin plate method for the demonstration of trypsin-like enzymes in cells are increased resolution and ability to show trypsin inhibitor effects.     

Further evidence for a possible role of trypsin-like activity in the adherence of Porphyromonas gingivalis.

The present study was undertaken to evaluate a possible correlation between the level of trypsin-like activity and the adherence properties of Porphyromonas gingivalis. It was demonstrated that strains with high cell-associated trypsin-like activity attach in higher numbers to human epithelial cells than strains with low levels of trypsin-like activity. To a lesser extent, the same tendency was also noted for the agglutination of human erythrocytes. The ability of P. gingivalis to attach to erythrocytes and epithelial cells was found to be affected by the presence of arginine and thiol protease inhibitors (leupeptin, p-chloromercuriphenylsulfonic acid). The inhibition profile was partially dependent on the age of the bacterial cells used in the adherence assay. It is suggested that adherence of mid-log P. gingivalis cells involves primarily trypsin-like proteases, whereas 2-day-old cells possess additional specific attachment mechanism(s).     

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

Haemin inhibits the trypsin-like enzyme activity of Porphyromonas gingivalis W83

The effect of haemin and related porphyrins on the activity of the trypsin-like enzyme activity in cell sonicates of Porphyromonas gingivalis was examined using a spectrophotometric assay and by following the degradation of human IgG. Haemin was inhibitory in both assay systems and the effect was shown to be reversible. The high concentration of haemin accumulated by P. gingivalis may protect the organism against autodegradative effects of the trypsin-like protease.     

The capture proteasome assay: A method to measure proteasome activity in vitro

Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i–β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome–Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO₄ in the proteasome–Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO₄ and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.     

Trypsin-like activity and thromboxane release in adult respiratory distress syndrome

Plasmatic immunoreactive trypsin (IRT), thromboxane and trypsin-like enzymatic activity were measured in 117 patients at risk of developing adult respiratory distress syndrome (ARDS) (53 multiple injury, 30 abdominal surgery, 17 acute pancreatitis, 12 burnt and 5 disseminated intravascular coagulation patients). 69 of these patients developed ARDS. Immunoreactive trypsin and thromboxane were measured by radio-immuno-assay and trypsin-like enzymatic activity by spectrophotometry, using a specific chromogenic substrate. Mean IRT value was 675 ng/ml in ARDS and 265 ng/ml in non ARDS patients (p less than 0.05). Mean IRT value was 685 ng/ml in septic and 170 ng/ml in non septic patients (p less than 0.01). An abnormal trypsin-like enzymatic activity was measured in 26 ARDS patients. In 60 patients (37 ARDS and 23 non ARDS), thromboxane appeared in plasma simultaneously or about 24 hours after the beginning of IRT release. The importance of thromboxane release parallels the intensity of IRT. Originating from pancreas, trypsin can appear in plasma either by absorption from gastrointestinal tract or after pancreatic ischemia.     

Trypsin-like activity and thromboxane release in adult respiratory distress syndrome

Plasmatic immunoreactive trypsin (IRT), thromboxane and trypsin-like enzymatic activity were measured in 117 patients at risk of developing adult respiratory distress syndrome (ARDS) (53 multiple injury, 30 abdominal surgery, 17 acute pancreatitis, 12 burnt and 5 disseminated intravascular coagulation patients). 69 of these patients developed ARDS.Immunoreactive trypsin and thromboxane were measured by radio-immuno-assay and trypsin-like enzymatic activity by spectrophotometry, using a specific chromogenic substrate.Mean IRT value was 675 ng/ml in ARDS and 265 ng/ml in non ARDS patients (p < 0.05). Mean IRT value was 685 ng/ml in sepatic and 170 ng/ml in non septic patients (p < 0.01). An abnormal trypsin-like enzymatic activity was measured in 26 ARDS patients. In 60 patients (37 ARDS and 23 non ARDS), thromboxane appeared in plasma simultaneously or about 24 hours after the beginning of IRT release. The importance of thromboxane release parallels the intensity of IRT. Originating from pancreas, trypsin can appear in plasma either by absortion from gastrointestinal tract or after pancreatic ischemia.     

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Proteinase activity in the early developmental stages of the carp, Cyprinus carpio

Embryonic and early postembryonic development of the carp is associated with an increase in the content of soluble proteins and nitrogen of non-protein nitrogenous components. Simultaneously, the activity of trypsin-like and chemotrypsin-like peptide hydrolases increases together with the increase in the degree of proteolysis. These data suggest that the development of carp embryos and early larva is accompanied by intensification of two opposite processes--synthesis and catabolism of proteins. The observed changes are more evident in early postembryonic period as compared with the early stages of embryogenesis.     

大豆胰蛋白酶抑制剂对棉铃虫幼虫消化生理和生长发育的影响

本研究根据棉铃虫Helicotverpa ormigera(Hubner)幼虫中肠蛋白酶在离体条件下对蛋白酶抑制剂的反应,选择具有较强抑制作用的大豆胰蛋白酶抑制剂,以0.21-4.2%(干重)的浓度配入幼虫人工饲料,测定了幼虫短期和长期取食这些饲料引起的中肠类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力的变化和生长抑制效应。短期取食抑制剂的幼虫,中肠弱碱性类胰蛋白酶活力显著增高,在4.2%。浓度下比对照高出21%;强碱性类胰蛋白酶、类胰凝乳蛋白酶和总蛋白酶活力显著降低,生长发育受到明显抑制。长期取食低浓度(0.84%)抑制剂的幼虫,弱碱性类胰蛋白酶和类胰凝乳蛋白酶活力显著增高,强碱性类胰蛋白酶活力显著降低。总蛋白酶活力变化不显著;长期取食高浓度(4.2%)抑制剂的幼虫,强碱性类胰蛋白酶和总蛋白酶活力显著降低,其它酶活力变化不显著。抑制剂随浓度的增高对幼虫生长的抑制作用加强,但浓度高于0.84%后,抑制强度的变化减小。据此作者认为,蛋白酶抑制剂对昆虫抗营养效应在于它对蛋白酶的激活和抑制作用,从而导致各种蛋白酶间的协调性破坏,昆虫消化过程受阻,影响生长发育。     

Chemical modification of the bovine pituitary multicatalytic proteinase complex by N-acetylimidazole. Reversible activation of casein hydrolysis

The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.     

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.     

Cry2Ab及Cry1Ac杀虫蛋白对棉铃虫中肠蛋白酶活性的影响

为明确Cry2Ab和Cry1Ac2种Bt杀虫蛋白单用与混用对棉铃虫Helicoverpa armigera（Htibner）中肠主要蛋白酶活性的影响,本文测定了取食含不同Bt蛋白人工饲料后棉铃虫中肠总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的差异。结果发现：Cry2Ab处理12h后对棉铃虫中肠总蛋白酶影响不大;对类胰蛋白酶的影响最大,除最高浓度处理外,其他浓度处理后棉铃虫类胰蛋白酶的活性明显高于对照;但对类胰凝乳蛋白酶活性的影响呈倒“V”字型,只有6．67ug／gCry2Ab处理后的棉铃虫酶活力显著高于对照,其他浓度处理与对照差异不显著或略低于对照;随着取食含Cry2Ab饲料时间的增加,棉铃虫中肠类胰蛋白酶和类胰凝乳蛋白酶的活性比对照显著增加;与对照相比,处理36h后类胰蛋白酶活性最高可增加到6．43倍。Cry1Ac处理棉铃虫12h后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性都明显增加,而且与处理浓度呈正相关;但是24h后,处理后棉铃虫的总蛋白酶和类胰凝乳蛋白酶活性明显降低,只有类胰蛋白酶活性仍高于对照,但活性增长倍数低于12h时的处理。Cru2Ab和Cry1Ac2种蛋白混用处理棉铃虫后,2种酶的酶活力基本低于Cry1Ac和Cry2Ab单用的酶活力之和;只有2种蛋白浓度均为2．22ug／g混用时,处理12h后类胰蛋白酶和类胰凝乳蛋白酶的活性高于2种蛋白单用时酶活力之和,且都显著的高于对照。     

Purification of alpha 2-macroglobulin with trypsin-like activity from pleural fluids.

An alpha 2-macroglobulin with trypsin-like activity has been purified from pleural fluids of patients suffering from chronic pancreatitis. The isolation procedure includes ammonium sulphate precipitation, gel-filtration on Sephadex G-200 and DEAE-cellulose chromatography. It gives 46-fold purification of alpha 2-macroglobulin with a 13% recovery. Based on titration experiments with pancreatic inhibitor, the protein from three different patients contained 0.28, 0.46 and 0.80 mol of trypsin-like protease per mol of alpha 2-macroglobulin.     

Protease activity associated with HeLa cell ribosomes

HeLa cells contain endoprotease activity, detected by a sensitive, solid phase assay. The endoprotease has the ability to cleave a variety of protein substrates and is trypsin-like in its sensitivity to inhibitors. The activity is in part associated with cellular ribosomes and polysomes. A variety of biological and physical-chemical treatments which alter ribosomes or protein synthesis also directly affect the ribosomal protease activity.